Methods and compounds for controlled release of recombinant parvovirus vectors

ABSTRACT

The invention uses recombinant parvoviruses, and particularly recombinant adeno-associated virus (rAAV) to deliver genes and DNA sequences for gene therapy following manipulation of the therapeutic virus for packaging and transport. The invention delivers therapeutic viral vectors via rAAV affixed to support matrixes (i.e., sutures, surgically implantable materials, grafts, and the like).

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/208,702, filed Jun. 1, 2000, the disclosure of whichis incorporated by reference herein in its entirety.

FEDERAL SUPPORT OF THE INVENTION

[0002] This invention was made with government support under grantnumber DK54419 from the National Institutes of Health. The United Statesgovernment has certain rights to this invention.

FIELD OF THE INVENTION

[0003] This invention relates to the controlled release of parvovirusvectors, and particularly the controlled in vivo release of recombinantadeno-associated (rAAV) viral vectors.

BACKGROUND OF THE INVENTION

[0004] The preparation and administration of many drugs and therapeuticproteins following freeze-drying (lyophilization) is known. Advances inpolymer chemistry and biomaterials have achieved the delivery of drugsvia attachment to graft materials. For example, heparin and urokinasebonding to vascular graft materials has been used in an attempt toimprove long-term patency of vascular grafts. Chemotherapy drugs havebeen impregnated into wafers for time-release delivery. None of thesesystems, however, deliver DNA with the goal of persistent therapeuticprotein expression in the host cells.

[0005] Known viral and non-viral (e.g. lipid-mediated,electroporation-enhanced) gene therapy delivery systems rely on thedelivery of gene vectors in solutions. The solutions may be injectedintravenously, intramuscularly, subcutaneously, or may even beadministered orally. These approaches are hampered by the fact that manygene therapy vectors or formulations are not stable (i.e., they lose theability to infect cells and deliver DNA) when the vector is exposed toalterations in conditions like temperature, hydration, or pH. Inaddition, these routes may lead to undesirably rapid biodistribution ofthe delivered virus to organs distant from the tissue of delivery (e.g.,within seconds after intravenous delivery, or within minutes to hoursafter intramuscular delivery). Accordingly, a need exists for methods inwhich gene delivery may be accomplished in a time-controlled or delayedmanner, and potentially with delayed biodistribution of virus distantfrom the target tissue.

[0006] Adeno-associated virus (AAV) is a nonpathogenic, helper-dependentmember of the parvovirus family. One of the identifying characteristicsof this group is the encapsidation of a single-stranded DNA (ssDNA)genome. The separate plus or minus polarity strands are packaged withequal frequency, and either is infectious. At each end of the ssDNAgenome, a palindromic terminal repeat (TR) structure base-pairs uponitself into a hairpin configuration. This serves as a primer forcellular DNA polymerase to synthesize the complementary strand afteruncoating in the host cell. Adeno-associated virus generally requires ahelper virus for a productive infection. Although adenovirus (Ad)usually serves this purpose, treatment of MV infected cells with UVirradiation or hydroxyurea (HU) will also allow limited replication.

[0007] Recombinant MV (rAAV) gene delivery vectors also package ssDNA ofplus or minus polarity, and must rely on cellular replication factorsfor synthesis of the complementary strand. While it was initiallyexpected that this step would be carried out spontaneously, by cellularDNA replication or repair pathways, this does not appear to be the case.Early work with rAAV vectors revealed that the ability to score markergene expression was dramatically enhanced when cells were co-infectedwith adenovirus, or transiently pretreated with genotoxic agents.Similar induction of rAAV vectors has been observed in vivo followingtreatment with Ad, ionizing radiation, or topoisomerase inhibitors.However, the effect was highly variable between different tissues andcell types.

[0008] The effort to establish the efficiency of AAV-mediated genedelivery following complete desiccation was described by Rabinowitz etal. in May, 1998 at the Third International Cancer Gene Therapy Meeting.At that time, it was shown that recombinant AAV remains infectious andcompetent for transduction (gene delivery leading to expression of thetransgenic protein) in a broad range of temperatures, pH, and hydrationstates. Since that time, the present inventors have performed additionalstudies of MV-mediated gene delivery following desiccation of the virus,based upon the reasoning that one limitation of gene therapy with viralvectors delivered to the bloodstream, or even delivered locally totissues like the lung or muscle in large doses, is virus scatter to manysites beyond the intended site of action.

[0009] One disadvantage of virus scatter is that generally, too littleof the therapeutic gene is present or delivered to the site where it isintended. For example, a CFTR gene intended to be delivered to the lungas a treatment for cystic fibrosis will have decreased utility if mostof the virus goes to the liver and spleen. Another problem with knowngene delivery systems is that it is difficult to judge an accuratedosage of the gene vector required to achieve the desired therapeuticeffect. Finally, if the viral vector has potentially toxic effects uponexposure to organs outside of the target, it is desirable to localizethe therapeutic treatment to the target only and decrease viral scatter.A persistent concern with gene therapy is the theoretical possibility ofunwanted gene delivery vector spreading to subjects' gonads, thusperhaps leading to insertional mutagenesis to germline cells.Accordingly, precise direction and amount of delivery of gene therapyvectors continues to be a priority in the development of safe andreliable gene delivery systems.

SUMMARY OF THE INVENTION

[0010] The invention uses recombinant parvoviruses, and in particular,recombinant adeno-associated virus (rAAV) to deliver nucleic acidsequences (i.e., genes and DNA sequences) for gene therapy following adehydration or drying step (i.e., partial or complete desiccation,lyophilization) in which the therapeutic virus vector is dried onto(i.e., affixed to) a support matrix. Useful support matrices includesurgically implantable materials (i.e., sutures, surgical graftmaterial, implantable devices and the like) for packaging and transportto a subject, thus allowing delivery of gene therapy via the rAAVaffixed to the support matrixes.

[0011] The present inventors have extensively characterized the use ofthe adeno-associated virus in solution as a gene transfer vector. Thepresent investigations characterizing the extraordinary stability ofrAAV as compared to other gene transfer viruses have led to thedevelopment of strategies to dehydrate the virus for purposes ofachieving stable transport of the virus, and drying rAAV ontoimplantable materials (e.g., sutures, graft materials and othersurgically implantable materials), in order to directly apply the virusto specific and local tissue sites for gene therapy.

[0012] The stability of rAAV over a range of conditions of hydration,pH, and temperatures, and solution content (e.g., amount of stabilizer,sugar, etc.) has been characterized. These studies led to thecharacterization of the stability of rAAV over time in a dehydratedstate. rAAV produced using the most commonly-utilized viral capsidstructure (AAV serotype 2) was found to maintain transduction potentialfollowing desiccation. rAAVs produced using serotype 1, 3, 4, and 5capsids were also found to maintain the potential for gene deliveryfollowing desiccation.

[0013] The present invention provides the ability to deliver genes viathe dehydrated virus when applied to various suture materials (silk, catgut, vicryl monofilament, and the like), to positively charged nylonmembranes, to a foam gelatin matrix, and to polytetrafluoroethylene(PTFE) graft materials. Gene delivery and protein expression have beendemonstrated using each of these materials in vitro and in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1A is a schematic illustrating the basic method of thepresent invention. A surgically implantable matrix such as a suture iscontacted with an MV vector, preferably an rAAV vector comprising aheterologous nucleic acid. The vector is then dried (i.e, dessicated)onto the surgically implantable matrix. As illustrated in FIG. 1B, thematrix comprising the desiccated AAV vector is then implanted orstitched into or onto a subject (e.g, a human or a non-human animal),preferably such that the matrix material is absorbed into the subjects'body. FIG. 1C is a graph illustrating that the percentage of hematocritin a mouse intramuscularly injected with a viral vector carryingerythropoietin (EPO) is lower over time than the percentage ofhematocrit in a mouse treated with sutures comprising the viral vectorcarrying EPO.

[0015]FIG. 2 is a graph of the embodiment of the invention described inExample 5 below. In this figure, “SS” mean “super suture,” which is asuture material treated with rAAV vector carrying the EPO gene. They-axis is labeled in terms of percentage hematocrit in thesubject.(here, a mouse). The x-axis is labeled in terms of days afterrAAV administration. The legend details which data points are fromintramuscular injection of the rAAV vector carrying the EPO gene, andwhich are from sutures onto which rAAV vector carrying the EPO gene hasbeen desiccated, and the dosage of vector administrated by each method.As shown in FIG. 1C, the percentage of hematocrit in a mouseintramuscularly injected with a viral vector carrying EPO is lower overtime than the percentage of hematocrit in a mouse treated with suturescomprising a viral vector carrying EPO.

[0016]FIG. 3 is a graphical illustration comparing the transduction ofrAAV/mEPO delivered intramuscularly and via rAAV/mEPO/suture delivery.

[0017]FIG. 4 is a graphical illustration of the successful delivery ofGFP gene and human factor IX genes with MV/Goretex® in tissue culture tocells.

[0018]FIG. 5 is a graphical illustration of a sample plot comparingperformance of rAAV serotypes 1, 2, 3, 4, and 5 following storage in thedried state in a buffer composed primarily of 25% dextrose solution.

[0019]FIG. 6 is a graphical illustration comparing the effect on rAAVType 2 stability of storage solutions comprising different stabilizersin varying concentrations.

DESCRIPTION OF THE PREF RR D EMBODIM NTS

[0020] The present invention will now be described more fullyhereinafter with reference to the accompanying drawings andspecification, in which preferred embodiments of the invention areshown. This invention may, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.

[0021] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. As used in thedescription of the invention and the appended claims, the singular forms“a”, “an” and “the” are intended to include the plural forms as well,unless the context clearly indicates otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

[0022] Except as otherwise indicated, standard methods known to thoseskilled in the art may be used for the construction of rAAV genomes,transcomplementing packaging vectors, and transiently and stablytransfected packaging cells for use in the present invention. Suchtechniques are known: to those skilled in the art. See e.g., J. Sambrooket al., Molecular Cloning: A Laboratory Manual Second Edition (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), and F. M.Ausubel et al., Current Protocols In Molecular Biology (Green PublishingAssociates, Inc. and Wiley-Interscience, New York, 1991).

[0023] Parvoviruses are relatively small DNA animal viruses thatcomprise a linear, single-stranded DNA genome. The term “parvovirus” asused herein encompasses the family Parvoviridae, includingautonomously-replicating parvoviruses and dependoviruses. The autonomousparvoviruses include members of the genera Parvovirus, Erythrovirus,Densovirus, Iteravirus, and Contravirus. The autonomous parvovirusesinclude members of the genera Parvovirus, Erythrovirus, Densovirus,Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include,but are not limited to, mouse minute virus, bovine parvovirus, canineparvovirus, chicken parvovirus, feline panleukopenia virus, felineparvovirus, goose parvovirus, and B19 virus. Other autonomousparvoviruses are known to those skilled in the art. See, e.g., K. I.Berns et al., “Parvoviridae: The Viruses And Their Replication,” inVirology, Third Edition (B. N. Fields et al., eds., Lippincott-Raven,Philadelphia, Pa., 1996, pp. 2173-2197).

[0024] Within the Parvovirus family, the genus Dependovirus contains theadeno-associated viruses (MV). Adeno-associated virus (MV) is anonpathogenic, helper-dependent member of the parvovirus family. Theadeno-associated viruses include, but are not limited to, MV type 1, MVtype 2, MV type 3, MV type 4, MV type 5, MV type 6, avian MV, bovine MV,canine MV, equine MV, and ovine AAV. In the use of the presentinvention, MV type 1, MV type 2, MV type 3, MV type 4, MV type 5 arepreferred, with AAV type 2 being particularly preferred.

[0025] Among the identifying characteristics of this group of viruses isa single-stranded DNA genome. In the case of MV, either the plus orminus polarity strand can be packaged with equal efficiency, and bothare infectious. Adeno-associated viruses generally require a helpervirus for a productive infection. Although adenovirus (Ad) usuallyserves this purpose, treatment of MV infected cells with UV irradiationor hydroxyurea will also allow limited replication.

[0026] Recombinant MV (rAAV) gene delivery vectors are also packaged assingle-strands of plus or minus polarity, and must rely on cellularreplication factors for synthesis of the complementary strand. While itwas originally expected that this step would be carried outspontaneously by cellular DNA repair synthesis pathways, this view nowappears to be over-simplified. Early work with rAAV vectors in culturedcells revealed that the ability to score marker gene expression wasdramatically enhanced when the cells were co-infected with adenovirus.This effect was observed in the absence of any AAV viral gene or knownAAV cis-acting transcriptional regulatory sequences. Two groupssubsequently demonstrated that the enhancement effect could be achievedthrough the expression of the Ad E4orf6 gene. Similar enhancement wasobserved when cells were treated with UV irradiation or other types ofcell stress. Further, the dosage of these treatments correlated with thelevel of enhancement, which also correlated with the conversion of thesingle-stranded virion DNA (vDNA) genome into duplex. Similar inductionof rAAV vectors has also been observed in vivo following treatment withAd, UV, ionizing radiation, or topoisomerase inhibitors. However, theeffect was highly variable between different tissues. More importantly,MV vectors in vivo have been defined as requiring one to two weeksbefore optimum levels of transgene expression can be observed, a featureascribed to second-strand synthesis See, e.g., Samulski et al.,“Adeno-associated Viral Vectors,” in The Development of Human GeneTherapy (T. Friedmann, ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1999).

[0027] The genomic sequences of various autonomous parvoviruses and thedifferent serotypes of AAV, as well as the sequences of the TRs, capsidsubunits, and Rep proteins are known in the art. Such sequences may befound in the literature or in public databases such as GenBank. See,e.g., GenBank Accession Numbers NC 002077, NC 001863, NC 001862, NC001829, NC 001729, NC 001701, NC 001510, NC 001401, AF063497, U89790,AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061,AH009962, AY028226, AY028223, NC 001358, NC 001540; the disclosures ofwhich are incorporated herein in their entirety. See also, e.g.,Chiorini et al., (1999) J. Virology 73:1309; Xiao et al., (1999) J.Virology 73:3994; Muramatsu et al., (1996) Virology 221:208;international patent publications WO 00/28061, WO 99/61601, WO 98/11244;U.S. Pat. No. 6,156,303; the disclosures of which are incorporatedherein in their entirety. An early description of the AAV1, AAV2 and MV3TR sequences is provided by Xiao, X., (1996), “Characterization ofAdeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

[0028] Preferably, the rAAV genome comprises (i.e., “carries”) aheterologous nucleic acid sequence, such as a DNA sequence or a gene.Any heterologous nucleotide sequence(s) may be delivered according tothe present invention. Nucleic acids of interest include nucleic acidsencoding peptides and proteins, preferably therapeutic (e.g., formedical or veterinary uses) or immunogenic (e.g., for vaccines) peptidesor proteins.

[0029] As used herein, the term “vector” or “gene delivery vector” mayrefer to a parvovirus (e.g., AAV) particle that functions as a genedelivery vehicle, and which comprises vDNA (i.e., the vector genome)packaged within a parvovirus (e.g., AAV) capsid. Alternatively, in somecontexts, the term “vector” may be used to refer to the vectorgenome/vDNA.

[0030] A “heterologous nucleotide sequence” will typically be a sequencethat is not naturally occurring in the virus. Alternatively, aheterologous nucleotide sequence may refer to a viral sequence that isplaced into a non-naturally occurring environment (e.g., by associationwith a promoter with which it is not naturally associated in the virus).

[0031] As used herein, a “recombinant parvovirus vector genome” is aparvovirus genome.(ie., vDNA) into which a heterologous (e.g., foreign)nucleotide sequence (e.g., transgene) has been inserted. A “recombinantparvovirus particle” comprises a recombinant parvovirus vector genomepackaged within a parvovirus capsid.

[0032] Likewise, a “rAAV vector genome” is an MV genome (i.e., vDNA)that comprises a heterologous nucleotide sequence. rAAV vectors requireonly the 145 base terminal repeats in cis to generate virus. All otherviral sequences are dispensable and may be supplied in trans (Muzyczka,(1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAVvector genome will only retain the minimal terminal repeat (TR)sequences so as to maximize the size of the transgene that can beefficiently packaged by the vector. A “rAAV particle” comprises a rAAVvector genome packaged within an MV capsid.

[0033] Parvovirus particles of the present invention may be “hybrid”particles in which the viral TRs and viral capsid are from differentparvoviruses. Preferably, the viral TRs and capsid are from differentserotypes of MV (e.g., as described in international patent publicationWO 00/28004, U.S. Provisional Application No. 60/248,920; U.S. patentapplication Ser. No. 09/438,268 to Rabinowitz et al., and Chao et al.,(2000) Molecular Therapy 2:619; the disclosures of which areincorporated herein in their entireties). Likewise, the parvovirus mayhave a “chimeric” capsid (e.g., containing sequences from differentparvoviruses, preferably different AAV serotypes) or a “targeted” capsid(e.g., a directed tropism) as described in international patentpublication WO 00/28004.

[0034] As used herein, a “parvovirus particle” encompasses hybrid,chimeric and targeted virus particles. Preferably, the parvovirusparticle has an MV capsid, which may further be a chimeric or targetedcapsid, as described above.

[0035] As used herein, the term “polypeptide” encompasses both peptidesand proteins, unless indicated otherwise.

[0036] As used herein, “transduction” or “infection” of a cell by MVmeans that the AAV enters the cell to establish a latent or active(i.e., lytic) infection, respectively. See, e.g., Virology, ThirdEdition (B. N. Fields et al., eds., Lippincott-Raven, Philadelphia, Pa.,1996, pp. 2173-2197). In embodiments of the invention in which a rAAVvector is introduced into a cell for the purpose of delivering anucleotide sequence to the cell, it is preferred that the MV integratesinto the genome and establishes a latent infection.

[0037] The viral capsid may be from any parvovirus, either an autonomousparvovirus or dependovirus, as described above. Preferably, the viralcapsid is an MV capsid (e.g., AAV1, AAV2, MV3, MV4, MV5 or AAV6 capsid).In general, the MV1 capsid, AAV5 capsid, and MV3 capsid are preferred.The choice of parvovirus capsid may be based on a number ofconsiderations as known in the art, e.g., the target cell type, thedesired level of expression, the nature of the heterologous nucleotidesequence to be expressed, issues related to viral production, and thelike. For example, the AAV1 capsid may be advantageously employed forskeletal muscle, liver and cells of the central nervous system (e.g.,brain); MV5 for cells in the airway and lung; MV3 for bone marrow cells;and AAV4 for particular cells in the brain (e.g., appendable cells).

[0038] In preferred embodiments, the parvovirus vector further comprisesa heterologous nucleotide sequence(s) (as described below) to bepackaged for delivery to a target cell. According to this particularembodiment, the heterologous nucleotide sequence is located between theviral TRs at either end of the substrate. In further preferredembodiments, the parvovirus (e.g., AAV) cap genes and parvovirus (e.g.,AAV) rep genes are deleted from the vector. This configuration maximizesthe size of the heterologous nucleic acid sequence(s) that can becarried by the parvovirus capsid.

[0039] Preferably, the heterologous nucleotide sequence or sequenceswill be less than about 5.0 kb in length (more preferably less thanabout 4.8 kb, still more preferably less than about 4.4 kb, yet morepreferably less than about 4.0 kb in length) to facilitate packaging bythe AAV capsid. Exemplary nucleotide sequences encode Factor IX,erythropoietin, superoxide dismutase, globin, leptin, thymidine kinase,catalase, tyrosine hydroxylase, as well as cytokines (e.g.,α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4,interleukin 12, granulocyte-macrophage colony stimulating factor,lymphotoxin, and the like), peptide growth factors and hormones (e.g.,somatotropin, insulin, insulin-like growth factors 1 and 2, plateletderived growth factor, epidermal growth factor, fibroblast growthfactor, nerve growth factor, neurotrophic factor -3 and -4,brain-derived neurotrophic factor, glial derived growth factor,transforming growth factor-α and -β, and the like).

[0040] The present invention may also be used to deliver vectors for thepurpose of expressing an immunogenic peptide or protein in a subject,e.g., for vaccination. The vectors will preferably comprise nucleic acidencoding any immunogen of interest known in the art including, but arenot limited to, immunogens from human immunodeficiency virus, influenzavirus, gag proteins, tumor antigens, cancer antigens, bacterialantigens, viral antigens, and the like.

[0041] As a further alternative, the heterologous nucleic acid sequencemay encode a reporter polypeptide (e.g., an enzyme such as GreenFluorescent Protein, alkaline phosphatase).

[0042] Alternatively, in particular embodiments of the invention, thenucleic acid of interest may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated trans-splicing (see, Puttaraju et al.,(1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No.6,083,702), interfering RNAs (RNAi) that mediate gene silencing (see,Sharp et al., (2000) Science 287:2431) or other non-translated RNAs,such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like.

[0043] The heterologous nucleic acid may be operably associated withexpression control elements, such as transcription/translation controlsignals, origins of replication, polyadenylation signals, and internalribosome entry sites (IRES), promoters, enhancers, and the like. Thoseskilled in the art will appreciate that a variety of promoter/enhancerelements may be used depending on the level and tissue-specificexpression desired. The promoter/enhancer may be constitutive orinducible, depending on the pattern of expression desired. Thepromoter/enhancer may be native or foreign and can be a natural or asynthetic sequence.

[0044] Promoters/enhancers that are native to the subject to be treatedare most preferred. Also preferred are promoters/enhancers that arenative to the heterologous nucleic acid sequence. The promoter/enhanceris chosen so that it will function in the target cell(s) of interest.Mammalian promoters/enhancers are also preferred.

[0045] Inducible expression control elements are preferred in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery are preferablytissue-specific promoter/enhancer elements, and include muscle specific(including cardiac, skeletal and/or smooth muscle), neural tissuespecific (including brain-specific), liver specific, bone marrowspecific, pancreatic specific, spleen specific, retinal specific, andlung specific promoter/enhancer elements. Other induciblepromoter/enhancer elements include hormone-inducible and metal-inducibleelements. Exemplary inducible promoters/enhancer elements include, butare not limited to, a tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametalothionein promoter.

[0046] In embodiments of the invention in which the heterologous nucleicacid sequence(s) will be transcribed and then translated in the targetcells, specific initiation signals are generally required for efficienttranslation of inserted protein coding sequences. These exogenoustranslational control sequences, which may include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

[0047] The vector may contain some or all of the parvovirus (e.g., MV)cap and rep genes. Preferably, however, some or all of the cap and repfunctions are provided in trans by introducing a packaging vector(s)encoding the capsid and/or Rep proteins into the cell. Most preferably,the vector does not encode the capsid or Rep proteins. Alternatively, apackaging cell line is used that is stably transformed to express thecap and/or rep genes (see, e.g., Gao et al., (1998) Human Gene Therapy9:2353; Inoue et a., (1998) J. Virol. 72:7024; U.S. Pat. No. 5,837,484;WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947).

[0048] A “therapeutic polypeptide” is a polypeptide that may alleviateor reduce symptoms that result from an absence or defect in a protein ina cell or subject. Alternatively, a “therapeutic polypeptide” is onethat otherwise confers a benefit to a subject, e.g., anti-cancer effectsor improvement in transplant survivability.

[0049] The parvovirus vector may also encode a heterologous nucleotidesequence that shares homology with and recombines with a locus on thehost chromosome. This approach may be utilized to correct a geneticdefect in the host cell.

[0050] The present invention may also be used to express an immunogenicpolypeptide in a subject, e.g., for vaccination. The nucleic acid mayencode any immunogen of interest known in the art including, but are notlimited to, immunogens from human immunodeficiency virus, influenzavirus, gag proteins, tumor antigens, cancer antigens, bacterialantigens, viral antigens, and the like.

[0051] The use of parvoviruses as vaccines is known in the art (see,e.g., Miyamura et al, (1994) Proc. Nat Acad. Sci USA 91:8507; U.S. Pat.No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et a.,U.S. Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al.; thedisclosures of which are incorporated herein in their entirety byreference). The antigen may be presented in the parvovirus capsid.Alternatively, the antigen may be expressed from a heterologous nucleicacid introduced into a recombinant vector genome. Any immunogen ofinterest may be provided by the parvovirus vector. Immunogens ofinterest are well-known in the art and include, but are not limited to,immunogens from human immunodeficiency virus, influenza virus, gagproteins, tumor antigens, cancer antigens, bacterial antigens, viralantigens, and the like.

[0052] An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GPI60 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol andenv genes products). The immunogen may also be an arenavirus immunogen(e.g., Lassa fever virus immunogen, such as the Lassa fever virusnucleocapsid protein gene and the Lassa fever envelope glycoproteingene), a poxyirus immunogen (e.g., vaccinia, such as the vaccinia L1 orL8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogenor a Japanese encephalitis virus immurogen), a filovirus immunogen(e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such asNP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFSviruses), or a coronavirus immunogen (e.g., an infectious humancoronavirus immunogen, such as the human coronavirus envelopeglycoprotein gene, or a porcine transmissible gastroenteritis virusimmunogen, or an avian infectious bronchitis virus immunogen). Theimmunogen may further be a polio immunogen, herpes antigen (e.g., CMV,EBV, HSV immunogens) mumps immunogen, measles immunogen, rubellaimmunogen, diptheria toxin or other diptheria immunogen, pertussisantigen, hepatitis (e.g., hepatitis A or hepatitis B) immunogen, or anyother vaccine immunogen known in the art.

[0053] Alternatively, the immunogen may be any tumor or cancer cellantigen. Preferably, the tumor or cancer antigen is expressed on thesurface of the cancer cell. Exemplary cancer and tumor cell antigens aredescribed in S.A. Rosenberg, (1999) Immunity 10:281). Other illustrativecancer and tumor antigens include, but are not limited to: BRCAI geneproduct, BRCA2 gene product, gp100, tyrosinase, GAGE-½, BAGE, RAGE,NY-ESO-1, CDK4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1,PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl.Acad. Sci. USA 91:3515); Kawakami et al., (1994) J. Exp. Med., 180:347);Kawakami et al., (1994) Cancer Res. 54:3124), including MART-1 (Coulieet al., (1991) J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J.Cutan. Pathol. 4:201) and MAGE antigen, MAGE-1, MAGE-2 and MAGE-3 (Vander Bruggen et al., (1991) Science, 254:1643); CEA, TRP-1, TRP-2, P-15and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neugene product (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin),TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA724, HCG, STN(sialyl Tn antigen), cerbB-2 proteins, PSA, L-CanAg, estrogen receptor,milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann.Rev. Biochem. 62:623); mucin antigens (international patent publicationWO 90/05142); telomerases; nuclear matrix proteins; prostatic acidphosphatase; papilloma virus antigens; and antigens associated with thefollowing cancers: melanomas, metastases, adenocarcinoma, thymoma,lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkinslymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer,prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidneycancer, pancreatic cancer and others (see, e.g., Rosenberg, (1996) Ann.Rev. Med. 47:481-91).

[0054] Accordingly, the present invention finds use in methods oftreating cancer or tumors, e.g., by delivery of anti-cancer agents orcancer antigens. In particular embodiments, the inventive methods areused to administer anticancer agents or cancer antigens to preventmetastasis, e.g., following surgical removal of a primary tumor.

[0055] The inventive methods and matrices may also advantageously beused in the treatment of individuals with metabolic disorders (e.g.,omithine transcarbamylase deficiency). Such disorders typically requirea relatively rapid onset of expression of a therapeutic polypeptide bythe gene delivery vector. As still a further alternative, the inventivevectors may be administered to provide agents that improve transplantsurvivability (e.g., superoxide dismutase) or combat sepsis.

[0056] rAAV vectors of the present invention are applied for deliveryonto surgically implantable matrices as described herein. In the methodof the present invention, rAAV vectors in solution are contacted withsurgically implantable matrices or materials and allowed to dry ordehydrate onto or into the surgically implantable materials or matrices.The vectors in solution may be contacted with the implantable matricesor materials by submerging or immersing the matrices or materials in thesolution. Alternatively, the vectors in solution may be contacted withthe implantable matrices in a drop-wise manner, or may be sprayed ontothe implantable matrices, or otherwise contacted with the matrices usingmethods that will be easily determined by those skilled in the art.

[0057] In a preferred embodiment of the invention, prior todrying/desiccation, the parvovirus (e.g., rAAV) vectors are in asolution and/or maintained in a solution (e.g., a storage solution)comprising a stabilizer. In other words, in a preferred embodiment, rAAVvectors are in a stabilizer-containing solution when they aredesiccated. It has been unexpectedly discovered by the present inventorsthat rAAV vectors in solutions comprising at least one stabilizer aremore stable after desiccation and rehydration/resuspension. Moreover,post-desiccation, the rAAV vectors originally desiccated from a solutioncomprising a stabilizer have a higher transduction efficiency whencompared to those vectors that were not desiccated from solutioncomprising a stabilizer (e.g., as compared to virus in PBS buffer only).Preferred stabilizers are sugars and sugar alcohols; more preferredstabilizers are sorbitol, sucrose, and dextrose, with sorbitol beingparticularly preferred. In preferred embodiments, the stabilizer (e.g.,sorbitol), comprises at least about 5% of the solution (e.g., is a 5%sorbitol solution); more preferably at least about 10% of the solution,even more preferably at least about 15%, still more preferably 20%, andin a most particularly preferred embodiment, at least 25%. In onepreferred embodiment, the storage solution comprising the vector/viruscomprises 25% sorbitol. FIG. 6 graphically illustrates the comparison ofstorage solutions comprising different stabilizers in varyingconcentrations; the comparison is expressed in terms of the stability ofrAAV 2 vectors (as a percentage of control vectors) seven days aftergene delivery and thirty days after gene delivery of the dried vector.

[0058] The drying or dehydrating step may be carried out according toknown techniques (desiccation, desiccation under vacuum conditions,lyophilization, evaporation, etc.). Desiccation or drying is preferablycomplete (i.e., at or approaching 0% solution or water), butalternatively may be partial.

[0059] Surgically implantable matrices or materials are those materialsthat may be inserted, implanted, sewn into, or otherwise contacted withthe cells, and/or tissues, and/or body of a subject, internally orexternally. Surgically implantable matrices or materials or the presentinvention may be porous or non-porous, flexible or non-flexible, weavedor non-weaved, absorbable or non-absorbable.

[0060] Preferably, the surgically implantable matrix is absorbable; thatis, the matrix is absorbed into the body of the subject over time. Suchmatrices include sutures, surgically implantable devices and materials,and graft materials. If absorption of the material and/or the rAAVvector is desired, the time or rate of absorption can be controlled byselection of material, by the dosage of the virus, location of theimplantable material, etc. Sutures used in the present invention may besewn onto or into skin, muscle, peritoneum or other surfaces in or onthe subjects' body. Surgically implantable materials (grafts, sutures,etc.) particularly useful in the present invention include vicryl andmonocryl (available from Ethicon, a subsidiary of Johnson and Johnson),NytranT™, Gortex® (polytetrafluoroethylene or “PTFE”), Marlex®,polypropylene, cat gut (chromic, about one to two weeks, until absorbedinto the body), polyglactin (three to four weeks for absorption), PDS(about six weeks for absorption), and Dacron®, with cat gut and Gortex®being presently preferred.

[0061] In one embodiment of the invention, the surgically implantablematerial is coated. Suitable coating materials are known in the art andinclude collagen and glutaraldehyde-linked collagen. Another strategythat has been used with grafts to be used inside blood vessels (wherethe development of fibrosis at the foreign implanted material is notdesired) is to bond heparin to the graft material that is releasedslowly over the first few weeks after the graft is implanted goes in.Albumin coatings have also been used, as well as certain coating gelsknown in the art. In one alternative embodiment of the invention,heparin is used to coat the surgically implantable material and used to“hold” or bind the rAAV to the implantable material (see U.S. patentapplication Ser. No. 09/228,203 to Samulski et al., the disclosure ofwhich is incorporated herein by reference in its entirety). The rAAV maythen be released from the implanted material by exchanging it withcellular receptors as the heparin is absorbed from the graft.

[0062] In other embodiments of the invention, helper virus functions areoptionally provided for the parvovirus vector, preferably at the site ofdelivery. In a preferred embodiment, the target tissue or cell of thegene delivery is treated with a helper function (i.e., is treated with asolution of helper virus) prior to the implantation of the rAAV/surgicalmatrix of the present invention. Both adenovirus and herpes simplexvirus may serve as helper viruses for MV. See, e.g., BERNARD N. FIELDSet al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-RavenPublishers). Exemplary helper viruses include, but are not limited to,Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barrvirus. The multiplicity of infection (MOI) and the duration of theinfection will depend on the type of virus used and the packaging cellline employed. Any suitable helper vector may be employed. Preferably,the helper vector(s) is a plasmid, for example, as described by Xiao etal., (1998) J. Virology 72:2224. The vector can be introduced into thepackaging cell by any suitable method known in the art, as describedabove.

[0063] A preferred method for providing helper functions employs anoninfectious adenovirus miniplasmid that carries all of the helpergenes required for efficient AAV production (Ferrari et al., (1997)Nature Med. 3:1295; Xiao et al., (1998) J. Virology 72:2224). The rAAVtiters obtained with adenovirus miniplasmids are forty-fold higher thanthose obtained with conventional methods of wild-type adenovirusinfection (Xiao et al., (1998) J. Virology 72:2224). This approachobviates the need to perform co-transfections with adenovirus (Holscheret al., (1994), J. Virology 68:7169; Clark et al, (1995) Hum. Gene Ther.6:1329; Trempe and Yang, (1993), in, Fifth Parvovirus Workshop, CrystalRiver, FL).

[0064] Herpesvirus may also be used as a helper virus for AAV. Hybridherpesviruses encoding the MV Rep protein(s) may advantageouslyfacilitate for more scalable AAV vector production schemes. A hybridherpes simples virus type I (HSV-1) vector expressing the AAV-2 rep andcap genes has been described (Conway et al., (1999) Gene Therapy 6:986and WO 00/17377, the disclosures of which are incorporated herein intheir entireties).

[0065] In view of the foregoing, the present invention is useful indelivering genes and other nucleic acid sequences either in vitro (tocells) or in vivo (to subjects) by contacting cells, tissues orsubjects' bodies to surgically implantable material treated with rAAV.Suitable subjects include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects are the most preferred. Human subjects include fetal,neonatal, infant, juvenile and adult subjects.

[0066] Recombinant virus vectors are preferably delivered to a targettissue in a biologically-effective amount. A “biologically-effective”amount of the virus vector is an amount that is sufficient to result ininfection (or transduction) and expression of the heterologous nucleicacid sequence in the target tissue. If the virus is administered in vivo(e.g., the virus is administered to a subject as described below), a“biologically-effective” amount of the virus vector is an amount that issufficient to result in transduction and expression of the heterologousnucleic acid sequence in a target cell.

[0067] The parvovirus vector administered to the subject may transduceany permissive cell or tissue. Suitable cells for transduction by theinventive parvovirus vectors include but are not limited to: neuralcells (including cells of the peripheral and central nervous systems, inparticular, brain cells), lung cells, retinal cells, epithelial cells(e.g., gut and respiratory epithelial cells), muscle cells, dendriticcells, pancreatic cells (including islet cells), hepatic cells,myocardial cells, bone cells (e.g., bone marrow stern cells),hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts,endothelial cells, prostate cells, germ cells, and the like.Alternatively, the cell may be any progenitor cell. As a furtheralternative, the cell can be a stem cell (e.g., neural stem cell, liverstem cell). As still a further alternative, the cell may be a cancer ortumor cell. Moreover, the cells can be from any species of origin, asindicated above.

[0068] Dosages of the inventive parvovirus particles will depend uponthe mode of administration, the disease or condition to be treated, theindividual subject's condition, the particular virus vector, and thegene to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁸-10¹³ transducing units,yet more preferably 10¹² transducing units.

[0069] The methods of the present invention also provide a means fordelivering heterologous nucleotide sequences into a broad range oftissue, cells, including tissue comprising dividing and/or non-dividingcells. The present invention may be employed to deliver a nucleotidesequence of interest to a cell in vitro, e.g., to produce a polypeptidein vitro or for ex vivo gene therapy. The cells, pharmaceuticalformulations, and methods of the present invention are additionallyuseful in a method of delivering a nucleotide sequence to a subject inneed thereof, e.g., to express an immunogenic or therapeuticpolypeptide. In this manner, the polypeptide may thus be produced invivo in the subject. The subject may be in need of the polypeptidebecause the subject has a deficiency of the polypeptide, or because theproduction of the polypeptide in the subject may impart some therapeuticeffect, as a method of treatment or otherwise, and as explained furtherbelow.

[0070] In general, the present invention may be employed to deliver anyforeign nucleic acid with a biological effect to treat or ameliorate thesymptoms associated with any disorder related to gene expression.Illustrative disease states include, but are not limited to: cysticfibrosis (and other diseases of the lung), hemophilia A, hemophilia B,thalassemia, anemia and other blood disorders, AIDS, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, epilepsy, and other neurological disorders, cancer, diabetesmellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher'sdisease, Hurlers disease, adenosine deaminase deficiency, glycogenstorage diseases and other metabolic defects, retinal degenerativediseases (and other diseases of the eye), diseases of solid organs(e.g., brain, liver, kidney, heart), and the like.

[0071] Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer could be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using antisensemutations. For unbalanced disease states, gene transfer could be used tocreate a disease state in a model system, which could then be used inefforts to counteract the disease state. Thus the methods of the presentinvention permit the treatment of genetic diseases. As used herein, adisease state is treated by partially or wholly remedying the deficiencyor imbalance that causes the disease or makes it more severe. The use ofsite-specific recombination of nucleic sequences to cause mutations orto correct defects is also possible.

[0072] The instant invention may also be employed to provide anantisense nucleic acid to a cell in vivo. Expression of the antisensenucleic acid in the target cell diminishes expression of a particularprotein by the cell. Accordingly, antisense nucleic acids may beadministered to decrease expression of a particular protein in a subjectin need thereof.

[0073] The application of desiccation/drying of rAAV for packaging andtransport and subsequent administration following reconstitution ordelivery on an implantable physical matrix is applicable in a broadrange of therapeutic situations. A clear application would be in vaccinedevelopment. rAAV vectors designed to provoke and maintain an immuneresponse (and innate host immunity, e.g. versus hepatitis orimmunodeficiency viruses) could be transported to developing countriesand delivered to even remote areas without need for stringentpreservation methods.

[0074] Additional embodiments and illustrations of the present inventioninclude the following:

[0075] rAAV-impregnated Gortex® or other graft material may be overlaiddirectly on muscle or other tissue to deliver to large muscle mass overtime. Other therapeutic strategies where this embodiment may beespecially effective include transducing a large, thin muscle like thediaphragm, where penetrating a large surface area of tissue could make adramatic difference in the pulmonary function, and using anti-cancergene delivery locally following surgical debulking of a tumor to killresidual tumor in the surgical bed (e.g. with angiostatin/endostatin).

[0076] Moreover, a large bulk of muscle can be transduced from thevascular bed with a strategy involving either rAAV-impregnated vasculargraft (or an rAAV vascular graft in combination with a bolusintra-vascular dose or viral vector). This embodiment involves isolatingthe artery that supplies, for example, the entire quadriceps muscle.Implanting an rAAV-impregnated graft into this artery causes virus to bereleased continuously into the entire end-capillary bed of the muscle,rather than simply the part of the muscle hit by an intramuscularinjection or the surface that a graft lies on. This would be analogousto injection directly into the portal vein, but the dose would notnecessarily be delivered all at once. In fact, the venous outflow of anindividual limb could be occluded temporarily (for a half hour orlonger) so that a bolus of virus could be delivered to saturateimmediate binding followed by the longer-term release from the graft asit is absorbed or heals in.

[0077] The present method would also be useful in AAV transduction invascular smooth muscle (i.e., coronary artery transduction/gancyclovirtreatment for restenosis).

[0078] The present invention finds particular use in the application oradministration of a known amount of rAAV virus as a film to permanent orabsorbable matrix for gene delivery to wide surface areas through whichdispersion would be difficult with injection methods. As an example, thehuman diaphragm muscle is several cell layers thick but a its totalsurface area is measured in square feet. Failure of the diaphragm muscleleads to the respiratory failure that ultimately contributes to death inthe disease muscular dystrophy. An implantable matrix material, capableof rapid off-loading of MV gene therapy vector (e.g. PTFE with amini-dystrophin gene), could be applied to the diaphragm muscle, andlead to uniformity of transduction over a large surface area in a waythat would be difficult to accomplish with multiple injections of genetherapy vector.

[0079] Similarly, sheets or films of rAAV may be directly and uniformlyadministered to flat or complex anatomic surfaces. For example, thepresent invention could be used to deliver rAAV vectors comprising orencoding antitumor agents directly into a tumor bed after complete orpartial resection, to prevent local tumor recurrences. It has beendemonstrated that a number of types of solid tumors will grow primarilyat the site of original tumor, but following resection of the originaltumor, distant sites of micro-metastatic disease will appear. Any of anumber of anti-neoplastic genes could be delivered this way, includinggene for angiogenesis inhibitor agents (e.g. endostatin (preferredembodiment),angiostatin), for direct tumor antimetabolites, and forinterleukins and other immune mediators to affect local or metastaticrecurrence.

[0080] In another embodiment, the invention facilitates the delivery ofgrowth factors onto absorbable or permanent tissue “scaffolds” in orderto direct remodeling of bone or soft tissue. For example, rAAV vectorsencoding genes for bone morphogenetic proteins (BMPs) or the upstreamfactors that mediate bone morphogenetic proteins could be stablyimmobilized onto biodegradable polymer scaffolds. The combined MVvirus/biomaterial can be used in a clinical setting as a bone-graftsubstitute to promote bone or ligament repair, as an osteo-inductive orchondrogenic mechanism. Synthetic materials that are currently used asstructural support for new bone formation include polylactic acidhomopolymers (PLA), plylactic acid-polyethylene glycol block copolymers(PLA-PEG), and polyglycolide (PGA). Candidate genes that could bedelivered via immobilized stably desiccated rAAV include growth factors(transforming growth factor-beta1), bone morphogenetic proteins (BMPs).As new bone or ligament responds to the rAAV-delivered growth factor andinfiltrates the scaffold, the scaffold can degrade and new bone orligament has been stimulated by the viral gene therapy to grow into andrepair the defect.

[0081] A similar strategy could be used to recruit and direct nerveregeneration. rAAV have already demonstrated the ability to delivergrowth factors in the central nervous system (e.g. glial cellline-derived neurotrophic factor GDNF). This and other nerve growthfactors NGFs could be localized to guide nerve regeneration by rAAV/NGFsthat have been desiccated onto inert implantable materials, e.g. forspinal cord regeneration.

[0082] In yet another embodiment of the invention vascular graftmaterials (e.g., GoreTex®) that have rAAV gene therapy vectors on thesurface or are impregnated with rAAV gene therapy vectors are developed.One problem of most vascular graft materials is that they tend tore-occlude due to ingrowth of endothelial cells (a process partiallydirected by angiogenic growth factors-preferred embodiment) andatherothrombotic processes. rAAV directing the expression of antisenseoligonucleotides against vascular endothelial growth factors, focaladhesion kinase (FAK), or a variety of other factors may allow prolongedvascular patency following vascular graft placement, particularly if thegene delivery can be controlled to occur at a slow off-rate over time.One embodiment is a vascular graft material for arterio-venoushemodialysis shunts, formulated with an rAAV vector that inhibits lossof patency of the shunt and an additional rAAV-regulated erythropoietinvector that corrects the erythropoietin-deficient anemia of renalfailure.

[0083] Alternatively, dried therapeutic rAAV may be formulated forslower release over time, following restoration of patency in coronary(heart) or peripheral blood vessels, delivering angiogenic growthfactors to reverse impaired cardiomyopathy or peripheral myopathy. Genedelivery to blood vessels has been attempted using transient completeocclusion of blood vessels and infusion of gene therapy vector, with thetime of exposure to the vector necessarily limited by the time the bloodvessel can be blocked. The ability to deliver the rAAV itself dried on avascular graft material with characterized properties of release of thevirus advantageously circumvents this limitation.

[0084] Several embodiments are suggested by the demonstrated stabilityof the rAAV when dehydrated from solutions of 10-25% stabilizer (e.g.,sorbitol), resulting in solid, buffered, sugar-based solid masses or“balls” of rAAV retaining transduction capability. For instance, it willbe possible to make beads of rAAV for directed local delivery oftherapy. For certain cancer treatment approaches, the present inventionfacilitates the release of rAAV from the sorbitol beads for moreprolonged exposure to rAAV vector than can be achieved using directinjection. More prolonged exposure will result in contact with a greaterproportion of cells that are cycling through the cell cycle, i.e. morecells that are susceptible to anti-neoplastic or suicide gene products.A strategy similar to the use of brachytherapy for intracavitaryradiation therapy could be applied.

[0085] Patients with cervical cancer are often treated withbrachytherapy, involving placement of afterloading applicators into thevaginal and uterine cavities and radiographic confirmation of properplacement of the applicators in proximity to the malignancy.Encapsulated radioactive sources are later inserted into theapplicators, and deliver anatomically localized radiation therapy overtime as the material (usually ¹³⁷CS or ²²⁶Ra) decays over days. Insteadof radiation, very localized, dried rAAV “balls” or “beads” could bepositioned in anatomic proximity to the lesion to deliver anticanceragents and/or achieve a “bystander” tumor kill that would not befeasible with simple injection of rAAV in solution. Such a strategy isfeasible for a range of cancers (currently treated with radiationbrachytherapy), including anal/rectal cancer, cervical carcinoma,endometrial carcinoma, vaginal cancer, pleural mesothelioma,nasopharyngeal carcinoma, and prostate cancer.

[0086] The demonstration of the stability of virus in thedried/desiccated state facilitates the delivery of molecular medicine,particularly in settings out side of tertiary care hospitals. Thestrategies developed for this application for stabilizing rAAV ofserotypes 1, 2, 3, 4 and 5 can also be applied to so-called chimeric orhybrid capsid structure rAAV vectors (see U.S. patent application Ser.No. 09/438,268, to Rabinowitz et al., incorporated herein by reference).

[0087] Currently, solutions of gene therapy vectors require carefulstorage conditions (generally requiring maintenance in frozen state at−20 or −80 degrees Celsius). The demonstration that rAAV vectors canmaintain efficient transduction capability dried at room temperaturemakes feasible delivery of molecular medicine to geographically ortechnologically remote areas, including to developing countries. Forinstance, an analogous application has been the development earlier inthis century of systems for maintaining viability of vaccines deliveredto developing countries. This system has depended on establishing a“cold chain” of refrigeration, which in some countries with unreliableelectricity has depended on kerosene or solar refrigeration. Theestablishment of efficacy of rAAV following room temperature storage inthe dried state, with the capacity to manipulate controlled release,suggest the present inventive strategies will be critical to thewide-spread development of rAAV vaccines. rAAV vectors designed toprovoke and maintain an immune response (and innate host immunity, e.g.versus hepatitis or humanimmunodeficiency viruses) could be transportedto developing countries and delivered to even remote areas without needto stringent preservation methods.

[0088] In general, gene therapy holds promise for correction of diseasestates for which available therapy for symptomatic treatment exists, butis too costly for the majority of the world's population. For example,the average cost of treatment with coagulation factor IX or VII for U.S.hemophilic patients averages $50,000-$100,000 United States dollars peryear. In underdeveloped countries, this cost precludes any treatment formost hemophilic patients, as does the need for refrigeration oftreatment products. Recent pre-clinical and clinical trials with rAAVsuggest that long-term correction of hemophilia with rAAV gene therapywill be achieved. The ability to prepare and transport rAAV as describedby this invention suggest that the limitations to use of conventionaltherapy in underdeveloped countries could be overcome by the stabilityof the desiccated/implantable rAAV.

[0089] Although applications for vaccine development or for the hostsecretion of a therapeutic protein (as in correction of hemophilia)generally lead to a systemic effect, one advantage of the presentinvention is the ability to deliver genes locally (i.e., notsystemically, but limited to a particular tissue, organ, area, etc.).For instance, following resection of a cancer of the type that is knownto recur locally, a cancer-fighting rAAV (i.e., an rAAV vectorcomprising a nucleic acid encoding an anti-cancer compound) on an inertmeshwork could be applied to the area of the tumor bed to prevent localrecurrence, as an adjuvant therapy for surgery and chemotherapy. Thelocalization of the therapy to the tumor bed and the potential localexpression of cancer-fighting genes could be more specific thanchemotherapy, which would either be administered systemically (witheffects on the whole body) or would rapidly diffuse away after localadministration. Diseases caused by protein deficiencies within specifictissue cell types, rather than by deficiencies of circulating proteins,could be targeted using an implanted matrix impregnated with the virusvector. For instance, delivery of the gene for dystrophin on a matrixapplied directly to the diaphragm muscle could lead to improvedpulmonary mechanics and decreased mortality in muscular dystrophy.

[0090] The following Examples are provided to illustrate the presentinvention, and should not be construed as limiting thereof.

EXAMPLE 1

[0091] Recombinant MV2 produced using a triple transfection protocol,and expressing the gene for the green fluorescent protein (GFP), wasapplied to 3.0 vicryl suture material (Ethicon, Somerville, N.J.) or toa circle of positively charged nylon membrane (GeneScreen Plus, NEN LifeScience, Boston,Mass.) and allowed to air dry in a room temperaturevacuum. HeLa cells were grown to approximately 80-90% confluence inmonolayer tissue culture were overlain with either the AAV/GFP/suture orthe MV/GFP/nytran and allowed to infect for periods of time up to 30minutes, prior to removal of the virus-impregnated material. Followingovernight incubation, cells were examined for transduction with GFP bydirect visualization of fluorescing cells using a Leitz DM IRBfluorescence microscopic (Leica, Heerbrugg, Switzerland).

[0092] Subsequently, rAAV/suture (coated 3-0 or 4-0 coated, braidedpolyglactin suture or 4-0 chromic gut suture) was prepared under sterileconditions in a negative pressure tissue culture hood by suspending 3.0vicryl suture material (Ethicon, Somerville, N.J.) across the lip ofautoclaved beaker and applying virus dropwise to the suture so that theexact titer of rAAV applied was known. Lengths of suture with knownapplied doses were then clipped and stored dry and sterile at roomtemperature for various lengths of time from days to months. Atsequential timepoints, lengths of rAAV/suture encoding the gene lacZwere applied to 293 cells in tissue culture, which had previouslyundergone a one hour incubation with adenovirus type 2. Following a onehour infection with suture/rAAVlacZ, the suture was removed. On thefollowing day, cells were stained for transduction and expression oflacZ, a marker gene that turns the cells blue under specific stainingconditions. Suture that had been prepared and stored at room temperatureas described did not lose appreciable transduction efficiency over thefirst week following preparation, and retained infectivity for at leastthree months.

EXAMPLE 2

[0093] In parallel to the experiments outlined in Example 1, Nytran®(positively charged nitrocellulose membrane) was investigated as aplatform for virus. Uniform-sized circles of Nytran® were prepared andsterilized by autoclaving. Various doses of virus (10⁶, 5×10⁵, 10⁵,5×10⁴, 10⁴ and 5×10³ transducing units) in a total volume of 10 ml wereadded to the membrane and dessicated in place under vacuum without heat.The underlining paper of the Nytran® disk was visibly wet by the virusin ten micro-liters. The Nytran® disks and the underlining paper wereplaced inverted on nearly confluent 293 cells, which were previouslyinfected with Ad (4 hrs prior to the matrix/rAAV). The position of thedisc on the plate was marked on the underside of the plate, and the diskremoved after one hour infection. The next day, the plates of cells werestained for lacZ and the blue staining observed to occur primarily inthe cells directly underlying the matrix/rAAV placement.

EXAMPLE 3

[0094] Each of the above experiments was repeated using rAAV/suture orrAAV/matrix carrying the gene for the marker green fluorescent protein(GFP) and performed on HeLa cells. Comparable results were obtained,with marker green fluorescence seen in cells that had been overlain withthe virus-impregnated materials.

EXAMPLE 4

[0095] Initial attempts at in vivo application of the rAAV/GFP/suturetechnology were carried out in mouse muscle, but were limited byautofluorescence in mouse muscle that rendered any detection ofGFP-mediated fluorescence above background fluorescence impossible. Toprove the utility of the inventive approach with a physiologicallyimportant gene, recombinant MV carrying the mouse gene forerythropoietin (rAAV/mEpo) was produced. Erythropoietin is normallysecreted by the kidneys and directs the bone marrow to increase redblood cell production, reflected in an increase in the hematocrit (thepercentage of blood volume occupied by red blood cells). rAAV/mEpo wasproduced using a triple plasmid transfection protocol which minimizesthe contamination of the rAAV prep with helper adenovirus proteins.Virus was further purified by centrifugation on an iodixinol gradientand FPLC column purification on a heparin sulfate column. Followingdetermination of rAAV vector genome titer by radioactive dot-blot assay,virus was dialyzed against sterile phosphate buffered saline (PBS) anddiluted to a concentration of 8×10¹¹ vector genomes/ml (vg/ml). Separatesutures prepared using the technique described above (suspension ofsuture with known volumes of virus aliquoted to suture in drop-wisefashion) with the following doses:

[0096] 2.4×10¹⁰ vg on single monofilament polyglactin (Monocryl® brandEthicon suture)

[0097] 7.2×10¹⁰ vg on single Monocryl suture

[0098] 9.6×10¹⁰ vg on single Monocryl suture

[0099] 1.9×10¹¹ vg on single Monocryl suture

[0100] 5×10¹¹ vg divided on one 5-0 chromic gut suture and one Monocrylsuture

[0101] Each suture was marked to delineate the length of suture on whichthe virus was located. Sutures were placed in a vacuum at lowtemperature with all virus being adsorbed within 24 hours.

EXAMPLE 5

[0102] Five 4 week-old BalbC mice had blood sampled to determinebaseline normal hematocrit levels. Hematocrit levels in all four micewere between 43 and 46%, at baseline. Each mouse was anesthetized withintraperitoneal Avertin and an incision made to expose the quadricepsmuscle of the leg. Suture was drawn through the muscle until the markerportion was positioned within the muscle bed, and tied in place. Theedges of the incision were reopposed, and the skin closed using one totwo skin staples. Retro-orbital blood samples were collected at ten dayintervals. By day twenty post-suture placement, the mouse receiving thehighest dose demonstrated a significantly higher hematocrit level thanmice receiving the lower dose, all of whom had levels comparable to anon-injected control mouse. This elevation continued for 105 dayspost-injection, at which time the highest-dose mouse was maintaining ahematocrit in the 65-74% range, with each of the other mice in the44-50% range (i.e., not significantly different from baseline or fromnon-injected control). The mice were sacrificed, and bone marrow fromthe polycythymic mouse prepared and analyzed by a hematopathologist, whoconfirmed supraphysiologic erythropoiesis in the bone marrow, consistentwith increased erythropoietin expression. These results are summarizedin FIG. 2.

EXAMPLE 6

[0103] Two additional experiments were conducted in order to support theresults set forth above. In one experiment, rAAV/mEpo virus in astandard preparation (dialyzed solution, stored in standard conditionsat −20° C., and not dessicated prior to use) was injectedintramuscularly in a wide range of doses into BalbC mice, and thereappeared to be a threshold dose for increase in hematocrit. Micereceiving doses of 8×10⁹ vg, 8×10¹⁰ vg performed similar to miceinjected with sterile saline or rAAV/lacZ virus, with no rise inhematocrit above baseline. Mice receiving 8×10¹¹ vg showed a markedincrease in hematocrit to at or above 80%. Therefore, the response ofthe supersuture mouse with the highest dose is consistent with theseintramuscular conditions and results. These results are graphicallyillustrated in FIG. 3.

[0104] Additionally, mice receiving intramuscular suture placement usingthe braided monofilament sutures impregnated with dessicated rAAV/lacZactually appeared to show a more marked non-specific scarring responsearound the suture placement, which appeared to negatively affectexpression.

EXAMPLE 7

[0105] Gortex® (PTFE) graft material was used as a platform for deliveryof dried MV. GFP gene and human factor IX genes were successfullydelivered to cells in tissue culture with the AAV/Gortex.

[0106] HeLa cells were plated at 1×10⁵ cells/well of 6-well plates. Allmedia used on cells was prepared with calf serum which had been preparedwith barium sulfate to remove any Vitamin K-dependent clotting factors,including bovine factor IX. Squares of polytetrafluoroethylene(PTFE)-coated vascular graft material (Gore-Tex®, GORE, Flagstaff,Arizona) cut to 0.5×0.5 cm or to 1.0×1.0 cm and autoclaved.

[0107] AAV2 prepared with a triple transfection protocol and purifiedwith iodixinol centrifugation and column chromatography was used, with aCMV promoter driving expression of the gene for human factor IX. Virusat doses to deliver 1×10⁴ virus vector genomes (vg)/cell or 1×10⁶vg/cell was applied to PTFE matrix. PTFE/MV overlain onto cells for 1hour, removed, and cells incubated overnight. On the day followinginfection, media was replaced with 1.5 ml of factor IX-depleted media.Twenty-four hours later, media was removed, aliquoted and factor IXsecreted into the media was quantitated using a sandwich ELISA withantibodies specific for human factor IX, as previously described(Monahan, et al., Gene Therapy 5, 40-49 (1998)). The comparison of thetwo AAV/matrix surface areas (0.25 cm² versus 1.0 cm²) versus virusapplied to the entire well (surface area 9.5 cm²) is expressed in FIG. 4as nanograms of human factor IX expressed per 24 hours.

[0108] These experiments suggested that the time for the virus tooffload from the surface of the Gortex® (teflon) is very rapid, andfurther suggest that different delivery platforms may be chosen fordifferent applications to achieve different timecourses anddistributions of gene transfer.

EXAMPLE 8

[0109] A preparation of rAAV2/murine erythropoietin cDNA virus that wasobtained using a method different from the foregoing was used: anidentical triple-transfection protocol was used for virus production,however purification of the rAAV was performed to isolate only the rAAVvirions that packaged a near-genome length strand of rAAV (so-called“self-complementary rAAV”). Pooled fractions of rAAV containingpredominantly self-complementing rAAV/mEpo virions were used to prepare5.0 absorbable gut suture or aliquoted as virus in solution to yield thesame total number of virus particles (=5×10¹⁰ vg) for infections.rAAV/mEpo suture.

[0110] Suture material was implanted or virus solution injectedintramuscularly into the right quadriceps muscle of Balb/cByJ mice underdirect visualization through a 4-5 mm skin incision. Control mice wereinjected with rAAV/LacZ virus (produced as noted above) in solution orafter preparation on suture in a manner identical to the mEpo virus.Sutures were prepared at least 48 hours prior to stitching into place.Whole blood was collected from the retro-orbital venous plexus at day 0and every 10 days thereafter. Hematocrit was determined bycentrifugation of blood in microcapillary tubes. These results indicatethat AAV vectors delivered on implantable materials can lead tolong-term functional gene expression.

EXAMPLE 9

[0111] AAV serotype 1, 2, 3, 4, and 5 virus vectors comprising GFP weredesiccated in a variety of potential stabilizing buffers includingsugars, salt, and/or proteins. Following seven days in the dried state,viruses were resuspended and used to infect cells in monolayer tissueculture. Transduction efficiency was compared to virus that had beenmaintained in solution on ice without freeze/thaws (i.e., compared aspercentage of control). A sample plot of the comparative performance ofrAAV serotypes 1, 2, 3, 4, and 5 following storage in the dried state ina buffer composed primarily of 25% dextrose solution is illustrated inFIG. 5.

[0112] The foregoing is illustrative of the present invention and is notto be construed as limiting thereof.

That which is claimed is:
 1. A method of delivering a viral vector to acell, comprising drying the vector onto a surgically implantable matrixand contacting a matrix with the cell.
 2. The method according to claim1 wherein the viral vector is a recombinant parvovirus vector.
 3. Themethod according to claim 1 wherein the viral vector is a recombinant MV(rAAV) vector.
 4. The method according to claim 3 wherein the rAAVvector is a recombinant MV Type 2 vector.
 5. The method according toclaim 3 wherein the rAAV vector is a recombinant MV Type 3 vector. 6.The method according to claim 3 wherein the rAAV vector is a recombinantMV Type 4 vector.
 7. The method according to claim 3 wherein the rAAVvector is a recombinant MV Type 5 vector.
 8. The method according toclaim 3 wherein the rAAV vector is a recombinant MV Type 1 vector. 9.The method according to claim 1, wherein the viral vector comprises aheterologous nucleic acid sequence.
 10. The method according to claim 1,wherein the viral vector is delivered in vitro.
 11. The method accordingto claim 1, wherein the viral vector is delivered to a subject in vivo.12. The method according to claim 11, wherein the subject is a humansubject.
 13. The method according to claim 1, wherein the surgicallyimplantable material is suture material.
 14. The method according toclaim 1, wherein the surgically implantable material is catgut.
 15. Themethod according to claim 1, wherein the surgically implantable materialis PTFE.
 16. The method according to claim 1, wherein the drying stepcomprises complete desiccation of the viral vector.
 17. The methodaccording to claim 1, wherein the vector is stored in a solution priorto drying.
 18. The method according to claim 17, wherein the solutioncomprises sorbitol.
 19. The method according to claim 17, wherein thesolution comprises at least 25% sorbitol.
 20. The method according toclaim 1, wherein the vector is a hybrid parvovirus vector.
 21. Themethod according to claim 1, wherein the vector is a chimeric parvovirusvector.
 22. The method of claim 9, wherein the heterologous nucleic acidcomprises nucleic acid encoding factor IX.
 23. A method of delivering aviral vector to a subject, comprising drying the viral vector onto asurgically implantable material, and implanting the material into thesubject.
 24. The method according to claim 23 wherein the viral vectoris a recombinant parvovirus vector.
 25. The method according to claim 23wherein the viral vector is a recombinant MV (rAAV) vector.
 26. Themethod according to claim 25 wherein the rAAV vector is a recombinant MVType 2 vector.
 27. The method according to claim 25 wherein the rAAVvector is a recombinant MV Type 3 vector.
 28. The method according toclaim 25 wherein the rAAV vector is a recombinant MV Type 4 vector. 29.The method according to claim 25 wherein the rAAV vector is arecombinant MV Type 5 vector.
 30. The method according to claim 25wherein the rAAV vector is a recombinant MV Type 1 vector.
 31. Themethod according to claim 23, wherein the viral vector comprises aheterologous nucleic acid sequence.
 32. The method according to claim23, wherein the subject is a human subject.
 33. The method according toclaim 23, wherein the surgically implantable material is suturematerial.
 34. The method according to claim 23, wherein the surgicallyimplantable material is catgut.
 35. The method according to claim 23,wherein the surgically implantable material is PTFE.
 36. The methodaccording to claim 23, wherein the drying step comprises completedesiccation of the viral vector.
 37. The method according to claim 23,wherein the vector is stored in a solution prior to drying.
 38. Themethod according to claim 37, wherein the solution comprises sorbitol.39. The method according to claim 37, wherein the solution comprises atleast 25% sorbitol.
 40. The method according to claim 23, wherein thevector is a hybrid parvovirus vector.
 41. The method according to claim23, wherein the vector is a chimeric parvovirus vector.
 42. The methodof claim 31, wherein the heterologous nucleic acid comprises nucleicacid encoding factor IX.
 43. A surgically implantable matrix, whereinrecombinant adeno-associated virus (rAAV) has been dried onto thesurgically implantable matrix.
 44. The surgical implantable matrixaccording to claim 43, wherein the rAAV vector is a recombinant MV Type2 vector.
 45. The surgical implantable matrix according to claim 43,wherein the rAAV vector is a recombinant MV Type 3 vector.
 46. Thesurgical implantable matrix according to claim 43, wherein the rAAVvector is a recombinant MV Type 4 vector.
 47. The surgical implantablematrix according to claim 43, wherein the rAAV vector is a recombinantAAV Type 5 vector.
 48. The surgical implantable matrix according toclaim 43, wherein the rAAV vector is a recombinant MV Type 1 vector. 49.The surgical implantable matrix according to claim 43, wherein the rAAVvector comprises a heterologous nucleic acid sequence.
 50. The surgicalimplantable matrix according to claim 49, wherein the heterologousnucleic acid sequence is a heterologous nucleic acid sequence encodingfactor IX gene.
 51. The surgical implantable matrix according to claim43, wherein the surgical matrix is selected from the group consisting ofsuture material, catgut and PTFE.
 52. The surgical implantable matrixaccording to claim 43, wherein the rAAV vector is stored in solutionprior to drying.
 53. The surgical implantable matrix according to claim52, wherein the solution comprises sorbitol.
 54. The surgicalimplantable matrix according to claim 52, wherein the solution comprises25% sorbitol.